Kuyesa kwa Hemoglobin Electrophoresis

Kuyesera Mfundo

Hemoglobin electrophoresis cholinga chake ndi kuzindikira ndi kutsimikizira hemoglobini osiyanasiyana abwinobwino komanso achilendo.

Chifukwa cha ma charger osiyanasiyana ndi ma isoelectric amitundu yosiyanasiyana ya hemoglobin, munjira ina ya pH buffer, pomwe hemoglobin ya isoelectric ili yotsika kuposa pH ya yankho la bafa, hemoglobin imanyamula chiwopsezo choyipa ndikusamukira ku anode panthawi ya electrophoresis. Mosiyana ndi zimenezi, hemoglobini yokhala ndi ndalama zabwino imasunthira ku cathode.

Pansi pa voteji inayake komanso pakadutsa nthawi inayake ya electrophoresis, ma hemoglobini okhala ndi ma charger osiyanasiyana ndi masikelo a molekyulu amawonetsa mayendedwe ndi liwiro losiyanasiyana. Izi zimalola kulekanitsa madera osiyana, ndipo kusanthula kotsatira kwa colorimetric kapena electrophoretic kutha kuchitidwa pazigawo izi kuti muwerenge ma hemoglobin osiyanasiyana. Njira yomwe imagwiritsidwa ntchito kwambiri ndi pH 8.6 cellulose acetate membrane electrophoresis.

Mkati mwa cytoplasm, magulu a ethylene glycol (CHOH-CHOH) omwe amapezeka mu glycogen kapena polysaccharide zinthu (monga mucopolysaccharides, mucoproteins, glycoproteins, glycolipids, etc.) ndi okosijeni ndi periodic acid ndikusandulika kukhala magulu a aldehyde (CHO-CHO). Magulu a aldehydewa amaphatikizana ndi mtundu wa Schiff reagent wopanda mtundu wa purplish-red, kupanga utoto wofiirira wofiyira womwe umayika pomwe ma polysaccharides amapezeka muselo. Izi zimatchedwa kuti periodic acid-Schiff (PAS) staining, yomwe kale imadziwika kuti glycogen staining.

Kuyesera Njira

Zida:Cellulose acetatemayichingwe, electrophoresis zipangizo(DYCP-38C ndi magetsi DYY-6C), Chida Chapamwamba Chotsitsa Zitsanzo (pipette), spectrophotometer, colorimetric cuvettes, zosungira.

Buffer:

(1) pH 8.6 TEB Buffer: Wezani 10.29 g Tris, 0.6 g EDTA, 3.2 g boric acid, ndi kuwonjezera madzi osungunuka ku 1000 ml.

(2) Borate Buffer: Yezani 6.87 g borax ndi 5.56 g boric acid, ndipo onjezerani madzi osungunuka ku 1000 ml.

Kachitidwe:

Pkukonzanso kwa Hemoglobin Solution

Tengani 3 ml ya magazi okhala ndi heparin kapena sodium citrate ngati anticoagulant. Centrifuge pa 2000 rpm kwa mphindi 10 ndikutaya madzi a m'magazi. Sambani maselo ofiira a magazi katatu ndi saline ya thupi (750 rpm, 5 minutes centrifugation nthawi iliyonse). Centrifuge pa 2200 rpm kwa mphindi 10 ndikutaya supernatant. Onjezani kuchuluka kofanana kwa madzi osungunuka, kenaka onjezerani nthawi 0,5 kuchuluka kwa carbon tetrachloride. Gwirani mwamphamvu kwa mphindi 5, kenako centrifuge pa 2200 rpm kwa mphindi 10 kuti mutenge yankho lapamwamba la Hb kuti mugwiritse ntchito pambuyo pake.

Kuyika Membrane

Dulani nembanemba ya cellulose acetate kukhala mizere yotalika 3 cm × 8 cm. Zilowetseni mu pH 8.6 TEB buffer mpaka zitakhuta, ndiye chotsani ndi kupukuta zouma ndi pepala losefera.

Kuwona

Gwiritsani ntchito chitoliro kuti muwone 10 μl ya hemoglobini yosungunuka pa cellulose acetate nembanemba (mbali yolimba), pafupifupi 1.5 cm kuchokera m'mphepete.

Electrophoresis

Thirani njira yothetsera borate mu chipinda cha electrophoresis. Ikani nembanemba ya cellulose acetate ndi mbali yamawanga kumapeto kwa cathode ya chipindacho. Kuthamanga pa 200 V kwa mphindi 30.

Elution

Dulani magawo a HbA ndi HbA2, kuwayika m'machubu osiyana oyesera, ndikuwonjezera 15 ml ndi 3 ml ya madzi osungunuka, motsatana. Pang'onopang'ono gwedezani kuti mutulutse hemoglobini kwathunthu, kenako sakanizani.

Colorimetry

Zero choyamwitsa pogwiritsa ntchito madzi osungunuka kuti athetse yankho ndikuyesa kuyamwa pa 415 nm.

Kuwerengera

HbA2(%) = Kutaya kwa chubu cha HbA2 / (Kuchotsedwa kwa chubu cha HbA × 5 + Kutuluka kwa chubu cha HbA2) × 100%

Kuwerengera Zotsatira Zoyeserera

Reference Range ya pH 8.6 TEB Buffer Cellulose Acetate Electrophoresis: HbA> 95%, HbA2 1% -3.1%

Zolemba

Nthawi ya electrophoresis siyenera kukhala yayitali kwambiri. Ma cellulose acetate membrane sayenera kuuma panthawi ya electrophoresis. Lekani electrophoresis pamene HbA ndi HbA2 asiyanitsidwa bwino. Kutalika kwa electrophoresis kungayambitse kufalikira kwa bandi ndi kusokoneza.

Pewani kugwiritsa ntchito zitsanzo zambiri. Kuchulukirachulukira kwamadzimadzi a hemoglobini kumatha kupangitsa kuti bandeti ikhale yotsekeka kapena kuti madontho osakwanira, zomwe zimapangitsa kuti ma HbA akweze mobodza.

Pewani kuipitsidwa kwa cellulose acetate nembanemba ndi mapuloteni.

Pakali pano sayenera kukhala okwera kwambiri; Apo ayi, magulu a hemoglobini sangalekana.

Nthawi zonse phatikizani zitsanzo za anthu abwinobwino komanso ma hemoglobini odziwika bwino monga zowongolera.

Beijing Liuyi Biotechnology imapanga tanki yaukadaulo ya electrophoresis ya hemoglobin electrophoresis yomwe ndi chitsanzo.DYCP-38Ccellulose acetate nembanemba electrophoresis thanki, ndipo pali mitundu iwiri ya magetsi electrophoresis kupezeka kwa cellulose acetate nembanemba electrophoresis thanki.DYY-2CndiDYY-6Cmagetsi.

Pakadali pano, Beijing Liuyi Biotechnology imapereka nembanemba ya cellulose acetate kwa makasitomala, ndipo kukula kwa cellulose acetate membrane kumatha kusinthidwa makonda. Takulandirani kuti mutifunse zitsanzo ndi zambiri.

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Tsopano tikuyang'ana mabwenzi, onse a OEM electrophoresis thanki ndi ogulitsa amalandiridwa.

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